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Title: Transient light-induced intracellular oxidation revealed by redox biosensor

Highlights: •Time-resolved live cell imaging revealed light-induced oxidation. •Only the roGFP probe fused with glutaredoxin reveals photooxidation. •The transient oxidation is rapidly reduced by the cytosolic antioxidant system. •Intracellular photooxidation is media-dependent. •Oxidation is triggered exclusively by exposure to short wavelength excitation. -- Abstract: We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30 s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.
Authors:
 [1] ;  [1] ;  [2] ;  [1] ;  [1] ;  [2] ;  [1] ;  [2] ;  [1] ;  [2] ;  [2] ;  [2]
  1. Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL 61801 (United States)
  2. (United States)
Publication Date:
OSTI Identifier:
22242121
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 439; Journal Issue: 4; Other Information: Copyright (c) 2013 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIOXIDANTS; DISULFIDES; EXCITATION; FLUORESCENCE; GLUTATHIONE; IMAGES; OXIDATION; REDOX POTENTIAL; THIOLS; VISIBLE RADIATION; WAVELENGTHS